Various cell culture techniques for limbal epithelial cells are currently being used for the transplantation of cultured limbal stem cells. In this study, we explored the possibility of using human limbal mesenchymal cells (HLMCs) as feeder layer for the human limbal epithelial cells (HLECs). Single cell suspension of HLECs was seeded onto denuded amniotic membranes with inactivated 3T3 fibroblasts or HLMCs as feeder layer. Expressions of Cytokeratin 3, Np63 and connexin 43 (Cx43) of the cultured epithelial cells were determined at 28 days and the ultrastructure of the epithelium was examined by transmission electron microscope after 14 days and 28 days of cultivation. In both groups, cells were differentiated into multilayer epithelium at 28 days. Basal cells of the cultured epithelium showed a strong nuclear labeling of Np63, but lacked CK3 and Cx43 expression. Transmission electron microscopy examination showed that there were abundant desmosomal contacts between the cells. The key feature the cultured epithelium was occurrence of a typical basement membrane. These results suggested that HLMCs can be used as an alternative feeder layer for HLECs, which makes the bioengineering product biologically safer for the clinical applications.
2009 John Wiley & Sons, Ltd.